-
Genes Jun 2024DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same...
DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.
Topics: Humans; Microsatellite Repeats; Real-Time Polymerase Chain Reaction; DNA Fingerprinting; Forensic Genetics; DNA
PubMed: 38927680
DOI: 10.3390/genes15060744 -
Antibiotics (Basel, Switzerland) May 2024The ultimate goal of antimicrobial stewardship (AMS) programmes is to decrease the occurrence and spread of antimicrobial resistance (AMR). In response to this, a...
The ultimate goal of antimicrobial stewardship (AMS) programmes is to decrease the occurrence and spread of antimicrobial resistance (AMR). In response to this, a pharmacist partnership was established between Malawi and Wales (UK) with the aim of strengthening antimicrobial stewardship (AMS) activities in Malawi, with the initial project focusing on two tertiary referral hospitals. The Global Point Prevalence Survey (GPPS) was undertaken for the first time in Malawi at these sites and demonstrated a prescribing rate slightly lower than the African average, with ceftriaxone indicated for almost every bacterial infection. An educational intervention was also delivered, with a train-the-trainer approach upskilling pharmacists at the two sites, who then cascaded co-produced training sessions to an additional 120 multidisciplinary health professionals. A toolkit to support AMS at an individual patient level was also developed and disseminated to provide an ongoing reference to refer to. Both the trainings and toolkit were well received. Over the course of this project, significant progress has been made with the AMS programmes at the two sites, with local staff empowered to implement AMS activities. These interventions could be easily replicated and scaled and support the delivery of some of the AMS elements of the Malawi Ministry of Health National Action Plan for Antimicrobial Resistance.
PubMed: 38927147
DOI: 10.3390/antibiotics13060480 -
Journal of Cachexia, Sarcopenia and... Jun 2024Aging negatively impacts tissue repair, particularly in skeletal muscle, where the regenerative capacity of muscle stem cells (MuSCs) diminishes with age. Although...
BACKGROUND
Aging negatively impacts tissue repair, particularly in skeletal muscle, where the regenerative capacity of muscle stem cells (MuSCs) diminishes with age. Although aerobic exercise is known to attenuate skeletal muscle atrophy, its specific impact on the regenerative and repair capacity of MuSCs remains unclear.
METHODS
Mice underwent moderate-intensity continuous training (MICT) from 9 months (aged + Ex-9M) or 20 months (aged + Ex-20M) to 25 months, with age-matched (aged) and adult controls. Histological examinations and MuSC transplantation assays assessed aerobic exercise effects on MuSC function and muscle regeneration. CCN2/connective tissue growth factor modulation (overexpression and knockdown) in MuSCs and AICAR supplementation effects were explored.
RESULTS
Aged mice displayed significantly reduced running duration (65.33 ± 4.32 vs. 161.9 ± 1.29 min, mean ± SD, P < 0.001) and distance (659.17 ± 103.64 vs. 3058.28 ± 46.26 m, P < 0.001) compared with adults. This reduction was accompanied by skeletal muscle weight loss and decreased myofiber cross-sectional area (CSA). However, MICT initiated at 9 or 20 months led to a marked increase in running duration (142.75 ± 3.14 and 133.86 ± 20.47 min, respectively, P < 0.001 compared with aged mice) and distance (2347.58 ± 145.11 and 2263 ± 643.87 m, respectively, P < 0.001). Additionally, MICT resulted in increased skeletal muscle weight and enhanced CSA. In a muscle injury model, aged mice exhibited fewer central nuclear fibres (CNFs; 266.35 ± 68.66/mm), while adult, aged + Ex-9M and aged + Ex-20M groups showed significantly higher CNF counts (610.82 ± 46.76, 513.42 ± 47.19 and 548.29 ± 71.82/mm, respectively; P < 0.001 compared with aged mice). MuSCs isolated from aged mice displayed increased CCN2 expression, which was effectively suppressed by MICT. Transplantation of MuSCs overexpressing CCN2 (Lenti-CCN2, Lenti-CON as control) into injured tibialis anterior muscle compromised regeneration capacity, resulting in significantly fewer CNFs in the Lenti-CCN2 group compared with Lenti-CON (488.07 ± 27.63 vs. 173.99 ± 14.28/mm, P < 0.001) at 7 days post-injury (dpi). Conversely, knockdown of CCN2 (Lenti-CCN2shR, Lenti-NegsiR as control) in aged MuSCs improved regeneration capacity, significantly increasing the CNF count from 254.5 ± 26.36 to 560.39 ± 48.71/mm. Lenti-CCN2 MuSCs also increased fibroblast proliferation and exacerbated skeletal muscle fibrosis, while knockdown of CCN2 in aged MuSCs mitigated this pattern. AICAR supplementation, mimicking exercise, replicated the beneficial effects of aerobic exercise by mitigating muscle weight decline, enhancing satellite cell activity and reducing fibrosis.
CONCLUSIONS
Aerobic exercise effectively reverses the decline in endurance capacity and mitigates muscle atrophy in aged mice. It inhibits CCN2 secretion from senescent MuSCs, thereby enhancing skeletal muscle regeneration and preventing fibrosis in aged mice. AICAR supplementation mimics the beneficial effects of aerobic exercise.
PubMed: 38925632
DOI: 10.1002/jcsm.13526 -
Proceedings of the National Academy of... Jul 2024Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome...
Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome instability, and cell death. Pathogenic bacteria encounter several genotoxic agents during infection. In keeping with this, the loss of DNA repair networks results in virulence attenuation in several bacterial species. Interstrand DNA crosslinks (ICLs) are a type of DNA lesion formed by covalent linkage of opposing DNA strands and are particularly toxic as they interfere with replication and transcription. Bacteria have evolved specialized DNA glycosylases that unhook ICLs, thereby initiating their repair. In this study, we describe AlkX, a DNA glycosylase encoded by the multidrug resistant pathogen . AlkX exhibits ICL unhooking activity similar to that of its homolog YcaQ. Interrogation of the in vivo role of AlkX revealed that its loss sensitizes cells to DNA crosslinking and impairs colonization of the lungs and dissemination to distal tissues during pneumonia. These results suggest that AlkX participates in pathogenesis and protects the bacterium from stress conditions encountered in vivo. Consistent with this, we found that acidic pH, an environment encountered during host colonization, results in DNA damage and that a is induced by, and contributes to, defense against acidic conditions. Collectively, these studies reveal functions for a recently described class of proteins encoded in a broad range of pathogenic bacterial species.
Topics: Acinetobacter baumannii; DNA Glycosylases; DNA Damage; DNA Repair; Acinetobacter Infections; Bacterial Proteins; Animals; Mice; DNA, Bacterial; Virulence; Escherichia coli
PubMed: 38923984
DOI: 10.1073/pnas.2402422121 -
Molecular Oncology Jun 2024Persistence of quiescent leukemia stem cells (LSCs) after treatment most likely contributes to chemotherapy resistance and poor prognosis of leukemia patients....
Persistence of quiescent leukemia stem cells (LSCs) after treatment most likely contributes to chemotherapy resistance and poor prognosis of leukemia patients. Identification of this quiescent cell population would facilitate eradicating LSCs. Here, using a cell-tracing PKH26 (PKH) dye that can be equally distributed to daughter cells following cell division in vivo, we identify a label-retaining slow-cycling leukemia cell population from AML1-ETO9a (AE9a) leukemic mice. We find that, compared with cells not maintaining PKH-staining, a higher proportion of PKH-retaining cells are in G0 phase, and PKH-retaining cells exhibit increased colony formation ability and leukemia initiation potential. In addition, PKH-retaining cells possess high chemo-resistance and are more likely to be localized to the endosteal bone marrow region. Based on the transcriptional signature, HLA class II histocompatibility antigen gamma chain (Cd74) is highly expressed in PKH-retaining leukemia cells. Furthermore, cell surface CD74 was identified to be highly expressed in LSCs of AE9a mice and CD34 human leukemia cells. Compared to LinCD74 leukemia cells, LinCD74 leukemia cells of AE9a mice exhibit higher stemness properties. Collectively, our findings reveal that the identified slow-cycling leukemia cell population represents an LSC population, and CD74 leukemia cells possess stemness properties, suggesting that CD74 is a candidate LSC surface marker.
PubMed: 38922758
DOI: 10.1002/1878-0261.13690 -
Virus Genes Jun 2024Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear...
Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.
PubMed: 38922563
DOI: 10.1007/s11262-024-02082-7 -
G3 (Bethesda, Md.) Jun 2024Multi-copied mitogenome are prone to mutation during replication often resulting in heteroplasmy. The derived variants in a cell, organ or an individual animal...
Multi-copied mitogenome are prone to mutation during replication often resulting in heteroplasmy. The derived variants in a cell, organ or an individual animal constitute a mitogene pool. The individual mitogene pool is initiated by a small fraction of the egg mitogene pool. However, the characteristics and relationship between them has not yet been investigated. This study quantitatively analyzed the heteroplasmy landscape, genetic loads, and selection strength of the mitogene pool of egg and hatchling in the silver carp (Hypophthalmichthys molitrix) using high-throughput resequencing. The results showed heteroplasmic sites distribute across the whole mitogenome in both eggs and hatchlings. The dominant substitution was Transversion in eggs and Transition in hatching accounting for 95.23% ± 2.07% and 85.38% ± 6.94% of total HP sites, respectively. The total genetic loads were 0.293 ± 0.044 in eggs and 0.228 ± 0.022 in hatchlings (p = 0.048). The dN/dS ratio was 58.03 ± 38.98 for eggs and 9.44 ± 3.93 for hatchlings (p = 0.037). These results suggest that the mitogenomes were under strong positive selection in eggs with tolerance to variants with deleterious effects, while the selection was positive but much weaker in hatchlings showing marked quality control. Based on these findings, we proposed a trans-generation dynamics model to explain differential development mode of the two mitogene pool between oocyte maturation and ontogenesis of offspring. This study sheds light on significance of mitogene pool for persistence of populations and subsequent integration in ecological studies and conservation practices.
PubMed: 38922124
DOI: 10.1093/g3journal/jkae101 -
Pathogens (Basel, Switzerland) Jun 2024MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an...
MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.
PubMed: 38921815
DOI: 10.3390/pathogens13060518 -
Asian Pacific Journal of Cancer... Jun 2024The study aimed to validate a method for minimizing phase errors by combining full-length lung 4DCT (f4DCT) scans with shorter tumor-restricted 4DCT (s4DCT) scans. It...
PURPOSE
The study aimed to validate a method for minimizing phase errors by combining full-length lung 4DCT (f4DCT) scans with shorter tumor-restricted 4DCT (s4DCT) scans. It assessed the feasibility of integrating two scans one covering the entire phantom length and the other focused on the tumor area. The study also evaluated the impact of Maximum Intensity Projection (MIP) volume and imaging dose for different slice thicknesses (2.5mm and 1.25mm) in both full-length and short target-restricted 4DCT scans.
METHODS
The study utilized the Quasar Programmable Respiratory Motion Phantom, simulating tumor motion with a variable lung insert. The setup included a tumor replica and a six-dot IR reflector marker on the breathing platform. The objective was to analyze volume differences in fMIP_2.5mm compared to sMIP_1.25mm within their respective 4D_MIP CT series. This involved varying breathing periods (2.5s, 3.0s, 4.0s, and 5.0s) and longitudinal tumor sizes (6mm, 8mm, and 10mm). The study also assessed exposure time and expected CTDIvol of s4D_2.5mm and s4D_1.25mm for different breathing periods (5.0s to 2.0s) in the sinusoidal wave motion of the six-dot marker on the breathing platform.
RESULTS
Conducting two consecutive 4DCT scans is viable for patients with challenging breathing patterns or when the initial lung tumor scan is in close proximity to the tumor location, eliminating the need for an additional full-length 4DCT. The analysis involves assessing MIP volume, imaging dose (CTDIvol), and exposure time. Longitudinal tumor shifts for 6mm are [16.6-17.2] in fMIP_2.5mm and [16.8-17.5] in sMIP_1.25mm, for 8mm [17.2-18.3] in fMIP_2.5mm and [17.8-18.4] in sMIP_1.25mm, and for 10mm [19-19.9] in fMIP_2.5mm and [19.4-20] in sMIP_1.25mm (p≥ 0.005), respectively.
CONCLUSION
The Quasar Programmable Respiratory Motion Phantom accurately replicated varied breathing patterns and tumor motions. Comprehensive analysis was facilitated through detailed manual segmentation of Internal Target Volumes and Internal Gross Target Volumes.
Topics: Humans; Four-Dimensional Computed Tomography; Phantoms, Imaging; Respiration; Feasibility Studies; Lung Neoplasms; Radiotherapy Planning, Computer-Assisted
PubMed: 38918671
DOI: 10.31557/APJCP.2024.25.6.2089 -
Nucleic Acids Research Jun 2024Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act...
Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.
PubMed: 38917328
DOI: 10.1093/nar/gkae526